The rat pineal gland possesses P2 receptors which potentiate the effect of noradrenaline-induced N′-acetyl-5-hydroxytryptamine ( N′-acetyl-5-HT) production. In the current study, this receptor was characterised according to agonist selectivity and signal transduction mechanisms. 2-MethylthioATP (2MeSATP), 2-chloroATP (2-ClATP), adenosine 5′- O-2-thiodiphosphate, (ADPβS), ATP and ADP, but not UTP, potentiated noradrenaline-induced N′-acetyl-5-HT production in a concentration-dependent manner. 2MeSATP neither induced the production of adenosine 3′:5′-cyclic monophosphate (cyclic AMP), nor inhibited its formation when the glands were stimulated by forskolin. The phospholipase C inhibitor 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U73122), but not the inactive analogue, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U73343), blocked the 2MeSATP effect. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-dissulphonic acid (PPADS), which inhibits phospholipase C-coupled P2Y 1 receptors, blocked the 2MeSATP effect. In conclusion, our data strongly suggest that the P2-like receptor that is present in rat pinealocytes and which is responsible for the potentiation of noradrenaline-induced N′-acetyl-5-HT production is a P2Y 1-like receptor, coupled to a G protein which stimulates phospholipase C.